Aberrant signaling by protein kinases is one of the driving forces of tumorigenesis. Transition from physiological to oncogenic processes is often triggered by changes in temporal and spatial regulation of kinases. Dissection of these events is limited by the capabilities of current tools. It remains difficult to manipulate activity of a specific kinase with precise timing and localization in living cells. To overcome current limitations we propose to develop a novel broadly applicable optogenetic tool that will allow us to regulate kinase activity in living cells using light. To control kinase activity in time and space we will engineer a novel light-sensitive allosteric switch based on fungal photoreceptor Vivid that changes conformation upon illumination with blue light. Insertion of the engineered switch at a specific site within the catalytic domain of a kinase will allow us to achieve light-mediated regulation of the activity. This will enable tightly controlled, reversible and localized regulation of a specific kinase in living cells. To demonstrate broad applicability of this tool we will use it for regulation of oncogenic protein kinases Src, Abl and PKA. To further expand application of this strategy we propose to develop light regulated PFKFB3, a structurally different kinase that phosphorylates fructose 6-phosphate to promote glucose metabolism in cancer cells. The reagents used in this method will be genetically encoded enabling ready application in many systems. Using light-mediated regulation of tyrosine kinase Src we will determine its novel role in regulation of signaling pathways that stimulate glucose metabolism during oncogenic transformation. We will employ light-controlled PFKFB3 to identify its role in localized regulation of glycolysis in different subcellular compartments of the cell and its effect on oncogenic morphodynamic changes and cell cycle.